Nucleic acid sequences encoding enantioselective amidases

ABSTRACT

The invention relates to nucleic acid sequence encoding enantioselective amides with an amino acid sequence of SEQ ID NO: 2. The invention also relates to a process for fermentation, comprising a batch and a feed phase, of a microorganism in a fermentation medium, wherein the microorganism expresses a nucleic acid according to the invention and wherein between 0.5 and 50 mg of Zn 2+ /ml of fermentation medium is fed during the fermentation. The invention also relates to a process for the preparation of enatiomerically enriched carboxylic acid and/or an enatiomerically enriched carboxylic acid amide, in which a mixture of corresponding D- and L-carboxylic acid amides is contacted with a expression product according to the invention in the presence of 0.01 mM–100 mM Zn 2+ , whereby one of the enantiomers of the carboxylic acid amide is enatiomerically hydrolysed to from corresponding enatiomerically enriched carboxylic acid, while the other enantiomer of the carboxylic acid amide remains unchanged.

CROSS-REFERENCE TO RELATED APPLICATION

This application is the national phase of PCT application PCT/NL02/00471having an international filing date of 15 Jul. 2002, which claimspriority from European applications 01202822.1 and 01202821.3, bothfiled 23 Jul. 2001. The contents of these documents are incorporatedherein by reference.

The invention relates to nucleic acid sequences encodingenantioselective amidases. The invention also relates to vectors, andhost cells comprising the nucleic acid sequences according to theinvention as well as processes for producing and using the expressionproducts of the nucleic acid sequences.

Amidases are polypeptides with amidase activity and are enzymes with theability to catalyze the hydrolysis of carboxylic acid amides to form thecorresponding carboxylic acids and ammonia. Enantioselective amidasesare amidases with a preference for one of the enantiomers of acarboxylic acid amide as a substrate. Enantioselective amidases areknown to be valuable in commercial bioprocesses in the production ofenantiomerically enriched carboxylic acids. Carboxylic acids are forexample α-H-α-amino acids, α,α-dialkylamino acids, α-hydroxy acidsand/or derivatives thereof as well as peptides thereof. Enantiomericallyenriched carboxylic acids-and/or derivatives thereof as well as peptidesare used in various industries, as for example the pharmaceuticalindustry, the agrochemical industry etc. For example, the amino acidL-valine is highly suitable as a precursor in cyclosporin Afermentations, α-hydroxypropionic acid is used in the production ofherbicides, some α-N-hydroxyamino acids can be used as anti-tumor agentsand D-p-hydroxyphenylglycine and D-phenylglycine are used in theproduction of certain semisynthetic broad-spectrum β-lactam antibiotics.In EP 494 716 B1, two examples of microorganisms displaying amidaseactivity are given: Ochrobactrum anthropi NCIB 40321 (also known asNCIMB 40321) and Klebsiella sp. NCIB 40322.

The invention specifically relates to nucleic acid sequences encodingenantioselective amidases with an amino acid sequence, which has atleast 70% identity with SEQ ID: NO. 2. In SEQ ID: NO. 1 the nucleic acidsequence encoding the L-amidase from Ochrobactrum anthropi NCIMB 40321is presented. In SEQ ID: NO. 2, the amino acid sequence corresponding tothe nucleic acid sequence of SEQ ID: NO. 1 is presented. It hassurprisingly been found that a fermentation, comprising a batch phaseand a feed phase, of a microorganism expressing a nucleic acid encodingan enantioselective amidase with an amino acid sequence, which has atleast 70% identity with SEQ ID: NO. 2 in a fermentation medium resultsin an increased production of enantioselective amidase activity if inthe feed phase between 0.5 and 50 mg/l fermentation medium Zn²⁺ is fedto the fermentation medium.

This increased production of enantioselective amidase activity isprobably due to an increase in activity of the enantioselective amidaseitself as well as to an increase in the amount of enantioselectiveamidase produced. In EP 1,174,499 A1 a nucleic acid sequence encoding anenantioselective amidase from Enterobacter cloacae N-7901 has beendisclosed; the corresponding amino acid sequence has 68% identity withthe amino acid sequence corresponding to the nucleic acid sequence ofSEQ ID NO: 1. The nucleic acid sequences encoding enantioselectiveamidases according to the invention are therefore new. The Zn²⁺dependency of the fermentation of the microorganism expressing thenucleic acid sequence encoding an enantioselective amidase ofEnterobacter cloacae N-7901 has not been described in EP 1,174,499.

The present invention preferably relates to nucleic acid sequencesencoding enantioselective amidases with amino acid sequences, which havea degree of identity with SEQ ID NO. 2 of at least about 75%, morepreferably at least about 80%, even more preferably at least about 85%,most preferably at least about 90%, more preferably at least 95% andeven more preferably at least 97%, in particular at least 98%, more inparticular at least 99%, most in particular 100%.

For purpose of the present invention, the degree of identity between twoamino acid sequences is determined by the BLASTP pairwise alignmentalgorithm (NCBl) with an identity table and the following alignmentparameters: Mismatch=−15, Penalty=−3, Gap-Extend=1, Match-Bonus=1, Gapx-droff=50, Expect=10, Word Size=3.

The present invention also relates to nucleic acid sequences encodingenantioselective amidases, which nucleic acid sequences preferablyhybridize under medium, more preferably under high stringency conditionsand most preferably very high stringency conditions with (i) SEQ ID NO.1, (ii) a genomic DNA sequence comprising SEQ ID NO.1 or (iii) acomplementary strand of (i) or (ii).

Hybridization experiments can be performed by a variety of methods,which are well available to the skilled man. General guidelines forchoosing among these various methods can be found in e.g. chapter 9 ofSambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: ALaboratory Manual. 2nd ed., Cold Spring Harbor Laboratory, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

With stringency of the hybridization conditions is meant, the conditionsunder which the hybridization, consisting of the actual hybridizationand wash steps, are performed. Wash steps are used to wash off thenucleic acids, which do not hybridize with the target nucleic acidimmobilized on for example a nitrocellulose filter. The stringency ofthe hybridization conditions can for example be changed by changing thesalt concentration of the wash solution and/or by changing thetemperature under which the wash step is performed (wash temperature).Stringency of the hybridization increases by lowering the saltconcentration in the wash solution or by raising the wash temperature.For purpose of this application, the hybridization is performed in 6×sodium chloride/sodium citrate (SSC) at about 45° C. for about 12 hours.Two consecutive 30 minutes wash steps in 1×SSC, 0.1% SDS at 50° C. is anexample of low stringency, at 55° C. an example of medium stringency, at60° C. an example of high stringency, at 65° C. an example of very highstringency.

The invention also relates to nucleic acid sequences encodingenantioselective amidases with an amino acid sequence according to SEQID: NO. 2 with alterations at about 15 or less amino acid positions,preferably at about 10 or less amino acid positions, more preferably atabout 5 or less, even more preferably at about 3 or less amino acidpositions, wherein the alteration(s) are/is independently (i) aninsertion of an amino acid (ii) a deletion of an amino acid (iii) asubstitution of an amino acid.

Nucleic acid sequences encoding enantioselective amidases with an aminoacid sequence given in SEQ ID: NO. 2 with a number of alterations, canbe prepared in a manner known in the art, for instance withsite-directed mutagenesis of the nucleic acid sequence. In this method amutagenic oligonucleotide encoding the desired mutation(s), such as asubstitution, insertion or deletion on a specific amino acid position,is annealed to one strand of the DNA of interest and serves as a primerfor initiation of DNA synthesis. By DNA synthesis a mutagenicoligonucleotide is incorporated into the newly synthesized strand.Often, the methods known in the art also have a positive selectiontechnique for mutagenic nucleic acid sequences in order to enhance theefficiency of the site-directed mutagenesis method. Some site-directedmutagenesis techniques make use of PCR, in which a mutagenicoligonucleotide is used as a primer. Methods for achieving site-directedmutations are described in various product folders of companies, as forexample Stratagene and Invitrogen and kits for achieving site-directedmutations are commercially available.

The present invention also relates to nucleic acid sequences encodingenantioselective amidases, which display immunological cross-reactivitywith an antibody raised against a fragment of the amino acid sequenceaccording to SEQ ID: NO. 2. The length of each fragment is preferably atleast 20 amino acids. The immunological cross reactivity may be assayedusing an antibody raised against, or reactive with, at least one epitopeof the isolated polypeptide according to the present invention havingamidase activity. The antibody, which may either be monoclonal orpolyclonal, may be produced by methods known in the art, e. g. asdescribed by Hudson et al., Practical Immunology, Third Edition (1989),Blackwell Scientific Publications. The immunochemical cross-reactivitymay be determined using assays known in the art, an example of which isWestern blotting, e. g. as described in Hudson et al., PracticalImmunology, Third Edition (1989), Blackwell Scientific Publications.

The present invention also relates to nucleic acid sequences encodingenantioselective amidase fusion proteins, which consist of a nucleicacid encoding a polypeptide according to the invention operativelylinked to one or more nucleic acid sequences, which encode (a) markerpolypeptide(s). With operatively linked is meant, that the two nucleicacid sequences are linked such that, if expressed, the enantioselectiveamidase fusion protein is produced with the marker polypeptide on its N-and/or C-terminus. The marker polypeptide can serve many purposes, forexample, it may be used to increase the stability or the solubility ofthe fusion protein, it may be used as a secretion signal, which is asignal that directs the fusion protein to a certain compartment in thecell or it may be used to facilitate purification of the fusion protein.Examples of marker polypeptides used to facilitate purification of thefusion protein are the MBP- and the GST-tag. The purification of afusion protein with a MBP-tag or a GST-tag is for example described inF. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J.A. Smith, and K. Struhl eds., Current Protocols in Molecular Biology,John Wiley & Sons, Inc., New York, N.Y., USA, 1990. A fusion proteinwith anMBP-tag can for example be produced in a pMAL vector (New EnglandBiolabs, Beverly, Mass., USA), whereas a fusion protein with a GST-tagcan be produced in a pGEX vector (Amersham Biosciences, Inc.,Piscataway, N.J., USA) by following the protocol of the respectivesupplier.

A nucleic acid sequence of the present invention, for example thenucleic acid sequence with the sequence of SEQ ID: NO. 1 can be isolatedusing standard molecular biology techniques and the sequence informationprovided herein. For example, using all or a portion of the nucleic acidsequence of SEQ ID: NO. 1 on Ochrobactrum anthropi NCIB 40321 as ahybridization probe, a nucleic acid sequence according to the inventioncan be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook, J., Fritsh, E. F., and Maniatis, T.Molecular Cloning: A Laboratory Manual 2nd, ed., Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989).

Moreover, a nucleic acid sequence encompassing all or a portion of SEQID: NO. 1 can be isolated by the polymerase chain reaction (PCR) usingsynthetic oligonucleotide primers designed based upon the sequenceinformation contained in SEQ ID: NO. 1 or SEQ ID: NO. 2, by using PCR onOchrobactrum anthropi NCIB 40321 and might also be isolated if theoligonucleotide primers are used on a microorganism displayingenantioselective amidase activity.

A nucleic acid sequence of the invention may also be amplified using forexample genomic DNA, cDNA or alternatively the appropriate mRNA from amicroorganism displaying enantioselective amidase activity, as atemplate and appropriate oligonucleotide primers based upon the sequenceinformation provided herein according to standard (RT)-PCR amplificationtechniques. The nucleic acid so amplified can be cloned into a suitablevector and characterized by DNA sequence analysis.

Furthermore, oligonucleotides corresponding to or hybridizable tonucleic acid sequences according to the invention can be prepared bystandard synthetic techniques, e.g., using an automated DNA synthesizer.

The nucleic acid sequences according to the invention can be cloned in asuitable vector and after introduction in a suitable host, the sequencecan be expressed to produce the corresponding enantioselective amidasesaccording to standard cloning and expression techniques, which are knownto the person skilled in the art (e. g., as described in Sambrook, J.,Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989). The invention also relates tosuch vectors comprising a nucleic acid sequence according to theinvention.

Suitable vectors are the vectors normally used for cloning andexpression and are known to the person skilled in the art. Examples ofsuitable vectors for expression in E. coli are given e.g. in table 1 inMakrides, S. C., Microbiological Reviews, Vol. 60, No. 3, (1996),512–538. Preferably, the vector contains a promoter upstream of thecloning site containing the nucleic acid sequence encoding thepolypeptide with amidase activity, which can be switched on after thehost has been grown to express the corresponding polypeptide havingamidase activity. Promoters, which can be switched on and off are knownto the person skilled in the art and are for example the lac promoter,the araBAD promoter, the T7 promoter, the trc promoter, the tac promoterand the trp promoter. Particularly useful in the framework of theinvention are for example the vectors as described in WO 00/66751, e.g.pKAFssECtrp or pKAFssECaro without the insert, the penicillin G acylasegene. Suitable hosts are the hosts normally used for cloning andexpression and are known to the person skilled in the art. Examples ofsuitable host strains are for example Echerichia coli strains, e.g. E.coli TOP10F′, TOP10, DH10B, DH5a, HB101, W3110, BL21(DE3) and BL21(DE3)pLysS. Particularly useful in the framework of the invention areEscherichia coli K-12 strains, e.g. DH1, HB101, RV308, RR1, W3110, C600.

The choice of the vector can sometimes depend on the choice of the hostand vice versa. If e.g. a vector with the araBAD promoter is being used,an E. coli host strain that is unable to break down the arabinoseinducer (ara-), is strongly preferred.

Alternatively, the nucleic acid sequences according to the invention canbe integrated into the genome of a host cell, which does not normallycontain a nucleic acid sequence according to the invention and be(over)expressed. This can be done according to methods known to theperson skilled in the art. The invention also relates to a host cell,which does not normally contain a nucleic acid sequence according to theinvention, comprising a nucleic acid sequence according to theinvention, preferably to a host cell comprising a vector comprising anucleic acid sequence according to the invention.

The invention also relates to a process for the preparation of theexpression product of a nucleic acid sequence according to any of claims1–5 wherein in a first step the nucleic acid sequence is introduced intoa suitable host, which does not normally contain a nucleic acid sequenceaccording to the invention, and wherein the nucleic acid sequence issubsequently expressed in said host. The introduction of a nucleic acidsequence and the subsequent expression are standard techniques known tothe person skilled in the art.

The invention also relates to a process for the fermentation, comprisinga batch and a feed phase, of a microorganism in a fermentation medium,wherein the microorganism expresses a nucleic acid according to theinvention and wherein between 0.5 and 50 mg/l_fermentation medium(corresponding to between 7.7 μM and 770 μM) Zn²⁺ is fed during thefermentation.

Typically, the feed phase is started after approximately 10 hours. Theamount of Zn²⁺ of 0.5–50 mg/l_fermentation medium can be fed at once tothe fermentation medium, but is preferably dosed, as the addition ofZn²⁺ at once gives rise to the formation of foam on the fermentationmedium and to lysis of the microorganism later on in the fermentation.Zn²⁺ can be dosed in for example 5–10 equal portions, but of course itis also possible to dose Zn²⁺ in different portions during the feedphase. Preferably, Zn²⁺ is continuously fed to the fermentation medium.If a continuous feed is used, it is very practical to combine Zn²⁺ withother components in one feed.

Zn²⁺ ions are for example present in zincsalts. In the process accordingto the invention preferably zinc salts that are well solvable in water(more than 0.1 moles per liter) are used, for example Zn(NO₃)₂,Zn(CH₃COO)₂, ZnSO₄, ZnCl₂, ZnBr₂, Znl₂.

The microorganism used in the fermentation can be a microorganism, whichpossesses and expresses a nucleic acid sequence according to theinvention by nature, but is preferably a host in which the nucleic acidsequence according to the invention is expressed, more preferablyoverexpressed.

It has been found that the enantioselective amidase activity of anexpression product of a nucleic acid sequence according to the inventionis also improved in the presence of between 0.01 mM and 100 mM Zn²⁺.Therefore, the invention also relates to a process for the preparationof an enantiomerically enriched carboxylic acid and/or anenantiomerically enriched carboxylic acid amide, in which a mixture ofthe corresponding D- and L-carboxylic acid amides is contacted with anexpression product according to any of claims 1–5 in the presence ofbetween 0.01 mM and 100 mM Zn²⁺, whereby one of the enantiomers of thecarboxylic acid amide is enantioselectively hydrolysed to form thecorresponding enantiomerically enriched carboxylic acid, while the otherenantiomer of the carboxylic acid amide remains unchanged. If desired,the remaining enantiomerically enriched carboxylic acid amide can behydrolysed to form the corresponding enantiomerically enriched acid. Thehydrolysis of the remaining carboxylic acid amide can be performed withmethods known in the art, for instance under basic or acidic conditions,or enzymatically.

Preferably, the enantioselective hydrolysis catalyzed by the expressionproduct of a nucleic acid sequence according to the invention isperformed in the presence of between 0.01 and 50, more preferablybetween 0.05 and 20 mM Zn²⁺.

The pH at which the enantioselective hydrolysis catalyzed by theexpression product of a nucleic acid sequence according to theinvention, takes place is not critical, preferably the enantioselectivehydrolysis is performed at a pH between 5 and 9, more preferably between6.5 and 8.5.

The temperature at which the enantioselective hydrolysis in the presenceof an expression product according to the invention takes place ispreferably between 10 and 75° C., more preferably between 30 and 65° C.,in particular between 40 and 60° C.

As a mixture, racemic mixtures of D- and L-carboxylic acid amide can beused, but of course it is also possible to use randomly chosen mixturesof D- and L-carboxylic acid amide.

Examples of suitable carboxylic acid amides are: α-H-α-amino acid amideswith 2–20 C-atoms or derivatives thereof, as for example alanine amide,phenylglycine amide, phenylalanine amide, para-hydroxyphenylglycineamide, proline amide, valine amide, leucine amide, tertiary leucineamide, methionine amide, proline amide, glutamic acid amide,α-H-α-hydroxy acid amides with 2–20 C-atoms, for example mandelic acidamide, α-α-dialkyl-amino acid amides with 2–20 C-atoms, for exampleα-methylvaline amide, α-methylphenylglycine amide, α-ethylphenylglycineamide, α-butylphenylglycine amide, α-methylphenylalanine amide,α-ethylphenylalanine amide, α-ethyl-α-butyl glycine amide. Preferablytert-leucine or α-methylphenylglycine is prepared in a process accordingto the invention.

DESCRIPTION OF THE DRAWINGS

FIG. 1: The relative L-amidase activity (rel.act.) of the samples fromfermentation with a feed 1 with Zn²⁺ (A) and with a feed 1 without Zn²⁺(B).

By way of illustration of the invention, the following examples havebeen added.

EXAMPLES Example 1 Fermentation of Escherichia coli K-12 Expressing theNucleic Acid Sequence Presented in SEQ ID: NO. 1 with the Addition ofZn²⁺ to the Fermentation Medium in the Feed Phase

The following seed medium was prepared:

Yeast extract powder (DIFCO, Bacto ™) 38 g Na₂HPO₄ 17.8 g KH₂PO₄ 13.6 gNH₄Cl 4.8 g Distilled water 1500 ml

The pH was adjusted to 6.8 with aqueous NaOH. 100 and 200 ml aliquotswere placed in 500 and 2000 ml Erlenmeyer flasks and sterilised (20 min.at 121° C.). 1.1 ml of a 50% (w/v) glucose and 2.2 ml of a neomycin (1.2g/l) solution were aseptically added to the 500 ml flask (seedphase 1).1 and 2 ml of the 50% (w/v) glucose and neomycin (1.2 g/l) solution wereadded to the 2000 ml flask (seedphase 2).

The Erlenmeyer flask of seedphase 1 was then seeded with a 1.8 ml 50%v/v glycerol/water suspension of the E. coli K-12 strain expressing thenucleic acid sequence according to SEQ ID: NO 1 and the whole wascultured and incubated for 22 hours at 27° C. under constant orbitalagitation. To express the nucleic acid sequence presented in SEQ ID NO.1, in the E. coli K-12 strain RV308, the nucleic acid sequence wasamplified by PCR using amidase specific primers with 5′-extensionscontaining a restriction site (NdeI for forward and SmaI for reverseprimer). The obtained fragment was cloned into a derivative of E. coliexpression vector pKECtrp at the site of E. coli penicillin G acylaseencoding sequence using restriction enzymes NdeI and SmaI. Thederivative expression vector used is similar to construct pKECtrp, whoseconstruction has been described in WO 00/66751, except that it containsthe E. coli aro promoter instead of the trp promoter. The wholeseedphase 1 Erlenmeyer flask was used to inoculate the 2000 mlErlenmeyer flask of the seedphase 2. Seedphase 2 was cultured andincubated for 3 hours at 27° C. under constant orbital agitation.

2% (v/v) of seedphase 2 was used to inoculate 6.4 l of batch phasemedium in a 20 l glass fermentor. The batch phase medium had thefollowing composition:

Gistex ® LS pasta  24.6 g/l Citric acid   10 g/l FeSO₄.7H₂O  0.2 g/lMgSO₄.7H₂O  3.1 g/l CaCl₂.2H₂O  1.5 g/l MnSO₄.H₂O 0.169 g/l (NH₄)₂SO₄ 9.0 g/l CoCl₂.6H₂O 0.006 g/l NaMoO₄.2H₂O 0.004 g/l H₃BO₃ 0.004 g/lAntifoam: Basildon 86/013 few drops K₂HPO₄ ¹  8.1 g/l Dextrose²  11.4g/l Neomycine² 0.012 g/l Thiamine² 0.014 g/l

The batch phase medium was sterilised at 121° C. for 65 minutes, afteradjusting the pH to 4.5 (with NaOH). The medium components marked with ¹and ² were each dissolved separately and the solution comprising thecomponents marked with ¹ were sterilised at 121° C. for 65 minutes andsolutions comprising the components marked with ² werefilter-sterilised. Both were added aseptically to the medium. Before thetransfer of the seedphase 1, the pH of the batch phase medium wasadjusted to pH 7.0. The fermentation was conducted at 27° C. underconstant stirring and optimal aeration conditions. Between 50 and 54hours, the temperature of the fermentation was linearly decreased during4 hours from 27 to 25° C. and maintained at that value till the end ofthe fermentation.

During the fermentation (batch and feed phase), the pH was allowed tovariate between 7.00 and 7.30. The batch phase was ended with the startof feed 1 and feed 2 (beginning of the feed phase) when the pH reachedthe level of 7.15. At that moment, the carbon source was exhausted. Thebatch phase had a duration of approximatively 9 hours. Feed 1 had thefollowing composition:

Dextrose 670 g/l Thiamine¹ 0.18 g/l MgSO₄.7H₂O¹ 14.4 g/l Proline¹ 6.1g/l Monosodiumglutamate¹ 12.2 g/l ZnSO₄.7H₂O¹ 0.022 g/l

Feed 1 was prepared as follows. The dextrose was first dissolved. About0.4 ml/l 4N HCl was added to the dextrose solution. The solution wassterilised (121° C. for 30 minutes). Thiamine, MgSO₄.7H₂O, proline andmonosodiumglutamate and ZnSO₄.7H₂O were dissolved separately and thesolution was filter-sterilised before addition (aseptically) to thedextrose solution. The profile used to introduce feed 1 into the batchphase medium is given below in table 1.

TABLE 1 Feed profile used to introduce feed 1. Time Setpoint[g_(feed)/(l_(start)*h)]  0 (Feedstart)–14 hours  2.59 × e^((0.07×Time))14–40 hours  6.91 + 0.48 × (Time − 14) 40 hours–end of fermentation 19.3

Feed 2 had the following composition:

Gistex® LS pasta 300 g/l

Feed 2 was prepared as follows. The yeast extract paste was firstdissolved in water. Subsequently, the pH was adjusted to 4.5+/−0.1. Thesolution was sterilised (121° C. for 30 minutes). The profile used tointroduce feed 2 into the batch phase medium is given below in table 2.With ‘Time’ in the profiles of table 1 and 2 is meant ‘hours afterfeedstart’.

TABLE 2 profile used to introduce feed 2. Time Setpoint[g_(feed)/(l_(start)*h)]  0 (Feedstart)–14 hours 0.81 × e^((0.07×Time))14–34 hours 2.15 + 0.15 × (Time − 14) 34–38 hours 5.15 38 hours–end offermentation 0

Feed 1 and feed 2 constitute the feed phase medium. The fermentation wasterminated after 120 hours.

The fermentation was repeated with the exception that in feed 1 noZnSO₄.7H₂O was present.

The L-amidase activity in the fermentation broth (fermentation mediumand cells) after start of the feed phase was determined for bothfermentations in the manner as described below:

Analysis of the L-amidase Activity of Fermentation Broth (FermentationMedium with Cells) Samples Obtained from the Fermentation of Escherichiacoli K-12 Expressing the Nucleic Acid Sequence Presented in SEQ ID: NO.1 with Zn²⁺ in Feed 1 and without Zn²⁺ in Feed 1

1.5 ml of incubation reagent (containing 1.1 w % L-phenylglycine amide,0.11 M HEPES-NaOH buffer, pH 8.0 and 1.1 mM MnSO₄.1H₂O) was heated in awaterbath to 55° C. After 10 minutes, 100 μl of a sample solution(containing fermentation broth diluted in 20 mM HEPES-NaOH, pH 7.5/2 mMDTT) was added to the heated incubation reagent. After incubation of thecombined incubation reagent with sample solution for 20 minutes, thereaction was stopped by adding 100 μl of the combined incubation reagentto 1.5 ml stop reagent (53 mM phosphoric acid). The stopped reactionmixture was centrifuged for 5 minutes at 14,000 rpm. The supernatant wasanalysed with HPLC under the following conditions:

-   column: nucleosil 120-3C18 (125×4 mm)-   wavelength detector: 220 nm-   flow: 1.0 ml/min-   injection volume: 20 μl-   eluent: 100 mM phosphate buffer pH 3.0-   Retention times of L-phenylglycine and L-phenylglycine amide are    respectively about 1.8 and 2.7 minutes.

The area under the HPLC peak corresponding to phenylglycine wascalculated and after correction for the dilution factor of thefermentation broth, compared to the area under the phenylglycine peakfrom other samples. The relative peak areas from the samples are equalto the relative L-amidase activity, which is the activity of the samplesas compared to one another. The relative L-amidase activity (rel. act.)of the samples from the fermentation with a feed 1 with Zn²⁺ (A) andwith a feed 1 without Zn²⁺ (B) is presented in FIG. 1, in which therelative activity has been plotted against T (h), the time (in hoursafter start of the batch phase) at which the samples were taken.

From FIG. 1, it can be seen that if Zn²⁺ is fed to the fermentation inthe feed phase, more L-amidase activity is produced.

Preparation of the Enzyme Solution LAM0011 from the Fermentation withZn²⁺ in Feed 1

The fermentation broth from the fermentation with Zn²⁺ in feed 1 wastreated with 4 g/l octanol in the fermentation to kill themicroorganisms and was homogenized twice by high pressure homogenization(600–700 bars). After homogenization the broth was flocculated byaddition of 10 v %, calculated on the volume of broth of a 10% solutionof C577. 10 v %, calculated on broth volume, of Dicalite 448, a filteraid, was added. The resulting slurry was filtered over a membranefilterpress and washed with 1 cake volume of process water. The biomasscake was sent to incineration. The filtrate was filtrated over Seitzfilterplates (sizes 5–15 μm) followed by 0.1–0.3 micrometer germfiltration plates. The resulting filtrate was stored in containers andfrozen until use.

Preparation of the Enzyme Solution LAM0001 from the Fermentation withoutZn²⁺ in Feed 1.

The fermentation broth from the fermentation without Zn²⁺ in feed 1 wastreated with 4 g/l octanol in the fermentation to kill themicroorganisms and was homogenized twice by high pressure homogenisation(600–700 bars). After homogenisation the broth was flocculated byaddition of 10% on the volume of broth of a 10% solution of C577. 10 v%, calculated on broth volume, of Dicalite 448, a filter aid, was added.The resulting slurry was filtered over a membrane filterpress and washedwith 2,2 cake volumes of process water. The biomass cake was sent toincineration. The filtrate was filtrated over Seitz filterplates sizes5–15 micrometer followed by 0.1–0.3 micrometer germ filtration plates.Hereafter the resulting filtrate was concentrated a factor 3 using 50 kDPolysulphon membranes. The retentate was washed with a quarter of theretentate volume of RO water. The retentate was hereafter againfiltrated over Seitz filterplates sizes 5–15 micrometer followed by0.1–0.3 micrometer germ filtration plates. The resulting filtrate wasstored into containers and frozen until use in the application reaction(examples 2–5).

Example 2 Enantioselective Hydrolysis of DL-α-methylphenylglycine Amidein the Presence of Different Concentrations of Zn²⁺ at pH 6.5 UsingL-amidase from Ochrobactrum anthropi NCIMB 40321

The effect of the Zn²⁺ concentration on the hydrolysis reaction ofDL-α-methylphenylglycine amide with the L-amidase from O. anthropi NCIMB40321, was determined by performing this reaction in the presence of0.1, 0.3, 1.0, 3.0 and 9.0 mM of Zn²⁺. As a control, a reaction withoutadditional Zn²⁺ was performed as well.

For this experiment, bottles were filled with 25 g of reaction mixture,each reaction mixture containing 2.5 g of DL-α-methylphenylglycine amide(end concentration 10 wt %), 46 μl from O. anthropi NCIMB 40321 batchno. LAM00101, and either no additional ZnSO₄ or 0.1, 0.3, 1.0, 3.0, or9.0 mM of ZnSO₄. The pH of all reaction mixtures was 6.5. The reactionswere started by the addition of the enzyme liquid.

All reaction mixtures were incubated on an orbital shaker at 55° C. and200 rpm. Directly after addition of the enzyme (t=0 hours) and after 1,4 and 11 hours samples of 1 g were taken and transferred to vialscontaining 4 g of 1 M H₃PO₄ to immediately stop the reaction. The exactamount of sample and stop solution were both determined by weighing.After filtration over a 0.22 μM filter, concentrations ofα-methylphenylglycine and α-methylphenylglycine amide were determined byHPLC according to the following protocol:

-   Column: Inertsil ODS-3 (3 μm) 50 mm*4.6 mm ID-   Eluent: 50 mM H₃PO₄—NaOH, pH=2.5-   Flow: 0.8 ml/min-   Temp.: 40° C.-   V_(inj.): 15 μL-   Detection: UV 210 nm

The results of this experiment are given in table 3, wherein theconversions (amount of amino acid formed as compared to the total amountof starting amino acid amide) of the reactions are given in relativeconversions; the highest conversion was set at 100.

TABLE 3 Influence of the Zn²⁺ concentration on the hydrolysis reactionof DL-α- methylphenylglycine amide by the L-amidase from O. anthropiNCIMB 40321 (batch no. LAM 0011). [Zn²⁺] Conversion after Entry (mM) 1 h4 h 11 h A 9.0 28 85 100 B 3.0 24 87 100 C 1.0 19 83 100 D 0.3 16 74 97E 0.1 11 61 92 F 0 1.4 3.1 5.3

From table 3 it can be concluded that the presence of Zn²⁺ is strangelypreferable for activity of the O. anthropi L-amidase towardsDL-α-methylphenylglycine amide. Without additional Zn²⁺, hardly anyhydrolysis reaction of this substrate occurs (see entry F).

Example 3 Enantioselective Hydrolysis of DL-α-methylphenylglycine Amidein the Presence of Different Divalent Metal Ions at pH 6.5 UsingL-amidase from Ochrobactrum anthropi NCIMB 40321

In a similar reaction set-up as for example 2, it was investigatedwhether additional Zn²⁺ had a beneficial effect on the conversion ofDL-α-methylphenylglycine amide with L-amidase from O. anthropi NCIMB40321, which had been fermented without an excess of Zn²⁺. Thehydrolysis reactions were performed in the presence of equimolar amountsof Mn²⁺ or Zn²⁺. Furthermore, a control reaction was performed using theL-amidase from O. anthropi NCIMB 40321 fermented in the presence of anexcess of Zn²⁺.

Bottles were filled with 25 g of reaction mixture, each reaction mixturecontaining 2.5 g of DL-α-methylphenylglycine amide (end concentration 10wt %), 240 μl of L-amidase from O. anthropi NCIMB 40321 preparationLAM0001, and 1 mM of either MnSO₄ or ZnSO₄. A third reaction mixture wasprepared containing (besides the substrate), 1 mM of Zn²⁺ and 92 μl ofL-amidase from O. anthropi NCIMB 40321 preparation LAM0011 (92 μlLAM0011 corresponds to the L-amidase activity of 240 μl LAM0001).Substrate incubation, sampling and analyses were all performed asdescribed in example 2. Results of this experiment are given in table 4,wherein the conversions (amount of amino acid formed as compared to thestarting amino acid amide) of the reactions are given in relativeconversions; the highest conversion was set at 100.

TABLE 4 Effect of the Zn²⁺ and Mn²⁺ addition on the hydrolysis reactionof DL-α-methylphenylglycine amide by the L-amidase from O. anthropiNCIMB 40321 fermented in the absence of Zn²⁺ in feed 1 (batch no. LAM0001) and fermented with Zn²⁺ in feed 1 (batch no. LAM 0011). ConversionEnzyme after Entry preparation Metal ion 1 h 3 h 5 h A LAM0001 Mn²⁺ 1133  55 B LAM0001 Zn²⁺ 33 94 100 C LAM0011 Zn²⁺ 36 93 100

From table 4 it can be concluded that in the presence of 1 mM of Zn²⁺,the rates of conversion of DL-α-methylphenylglycine amide with theenzymes fermented with and without extra Zn²⁺ are similar and are bothhigher than the conversion in the presence of 1 mM Mn²⁺.

Example 4 Enantioselective Hydrolysis of DL-tert-leucine Amide in thePresence of Different Divalent Metal Ions at pH 7.0 by L-amidase fromOchrobactrum anthropi NCIMB 40321

In a similar set-up as for example 2, the effect was determined of thepresence of equimolar amounts of Mn²⁺, Zn²⁺, and Mg²⁺ in the hydrolysisreaction of DL-tert-leucine amide with the L-amidase from O. anthropiNCIMB 40321. As a control, a reaction without any additional divalentmetal ion was performed.

To perform this experiment, bottles were filled with 25 g of reactionmixture, each reaction mixture containing 3.13 g of DL-tert-leucineamide (end concentration 12.5 wt %), 1.45 ml of L-amidase from O.anthropi NCIMB 40321 (batch no. LAM0011), and 1.0 mM of ZnSO₄, MgSO₄ orMnSO₄. Furthermore, a separate reaction was performed without anyadditional divalent metal ion. The pH of all reaction mixtures was 7.0.The reactions were started by the addition of the enzyme liquid.

All reaction mixtures were incubated on an orbital shaker at 55° C. and150 rpm. Directly after addition of the enzyme (t=0 hours) and after 1,3 and 6.7 hours samples of 1 g were taken and transferred to vialscontaining 4 g of 1 M H₃PO₄ to immediately stop the reaction. Both theexact amount of sample and stop solution were determined by weighing.After filtration over a 0.22 μM filter, concentrations of tert-leucineand tert-leucine amide were determined by HPLC according to thefollowing protocol:

-   Column: Inertsil ODS-3 (150 mm×4.6 mm I.D., 5μ), supplied by    Varian-Chrompack-   Eluent: 99 v/v % 50 mM H₃PO₄, pH=2.3 using 1N NaOH+1 v/v %    acetonitrile-   Flow: 1.0 ml/min-   Temp.: 30° C.-   V_(inj.): 20 μl-   Detection: fluorescence detection after post-column derivatization    with OPA/MCE^((a)) (λ_(ex) 365 nm and λ_(em)>420 nm).-    ^((a)) post-column derivatization was performed in a reaction coil    (2 m×0.25 mm I.D.) at ambient temperature using 0.4 M borate buffer    pH 10 containing o-phthalaldehyde (OPA) and 2-mercaptoethanol (MCE).    The flow of the derivatization reagent was 1.0 ml/min. The reagent    was prepared by first dissolving 49.46 g H₃BO₃ and 35 g KOH in 2    liter Milli-Q water, followed by adjustment of the pH to 10 using 1M    KOH. Then 1.6 g OPA (in 20 ml ethanol) and 2 ml MCE were added to    this borate buffer.

The results of this experiment are given in table 5, wherein theconversions (the amount of amino acid formed as compared to the amountof starting amino acid amide) of the reactions are given in relativeconversions, the highest conversion was set at 100.

TABLE 5 Influence of the presence of different divalent metal ions onthe hydrolysis reaction of DL-tert-leucine amide by the L-amidase fromO. anthropi NCIMB 40321 (batch no. LAM 0011). Conversion after EntryMetal ion 1 h 3 h 6.7 h A Zn²⁺ 15 56 100 B Mg²⁺ 8.9 32 77 C none 8.1 3069

The data in table 5 clearly show that in the presence of 1 mM of Zn²⁺ afaster hydrolysis reaction of DL-tert-leucine amide by the L-amidasefrom O. anthropi NCIMB 40321 is obtained than in the presence of anequimolar amount of Mg²⁺ or in the absence of an additional divalentmetal ion.

Example 5 Enantioselective Hydrolysis of DL-tert-leucine Amide in thePresence of Zn²⁺ at Different pH Values with L-amidase from Ochrobactrumanthropi NCIMB 40321

To investigate the influence of pH on the activity of L-amidase from O.anthropi NCIMB 40321 on the enantioselective hydrolysis ofDL-tert-leucine amide, reactions in the presence and absence of 1 mMZn²⁺ were performed at pH 7.0, 8.0 and 9.0.

The whole experimental set-up was identical to the set-up of example 3.The results of these experiments are presented in table 6.

TABLE 6 Influence of the pH of the reaction mixture on the hydrolysisreaction of DL-tert-leucine amide in the presence and absence of 1 mMZnSO₄ by the L-amidase from O. anthropi NCIMB 40321 (batch no. LAM0011). Zn²⁺ Conversion after Entry pH (1 mM) 1 h 3 h 6.7 h A 7 + 15 56100 B − 8.1 30 69 C 8 + 14 46 94 D − 11 38 82 E 9 + 14 43 93 F − 13 3977

1. An isolated nucleic acid molecule comprising a nucleotide sequenceencoding an enantioselective amidase wherein said amidase has an aminoacid sequence that has at least 95% identity with SEQ ID NO:2.
 2. Thenucleic acid molecule of claim 1, wherein said amino acid sequence isidentical to SEQ ID NO:2.
 3. The nucleic acid molecule of claim 1,wherein said encoding nucleotide sequence is operatively linked to oneor more nucleotide sequences, which encode (a) marker polypeptide(s). 4.A vector comprising the nucleic acid molecule according to claim
 1. 5. Amicroorganism modified to contain the nucleic acid molecule according toclaim
 1. 6. A process for the preparation of an enantioselectiveamidase, wherein said process comprises culturing the microorganism ofclaim 5 and isolating said enantioselective amidase.
 7. A process forthe fermentation of a microorganism in a fermentation medium, whereinsaid process comprises culturing said microorganism in a batch and afeed phase, wherein said microorganism expresses a nucleic acid sequenceaccording to claim 1 and wherein between 0.5 and 50 mg/l Zn²⁺ is fed perliter fermentation medium during the feed phase of the fermentation. 8.A process for the preparation of an enantiomerically enriched carboxylicacid and/or an enantiomerically enriched carboxylic acid amide, whichcomprises contacting a mixture of the corresponding D- and L-carboxylicacid amides with the amidase encoded by the nucleic acid moleculeaccording to claim 1 in the presence of between 0.01 mM and 100 mM Zn²⁺,whereby one of the enantiomers of the carboxylic acid amide isenantioselectively hydrolysed to form the corresponding enantiomericallyenriched carboxylic acid, while the other enantiomer of the carboxylicacid amide remains unchanged.
 9. An enantioselective amidase prepared bythe process of claim
 6. 10. An enantioselective amidase prepared by theprocess of claim
 7. 11. A process for the preparation of anenantiomerically enriched carboxylic acid and/or an enantiomericallyenriched carboxylic acid amide, which comprises contacting a mixture ofthe corresponding D- and L-carboxylic acid amides with the amidase ofclaim 9 in the presence of between 0.01 mM and 100 mM Zn²⁺, whereby oneof the enantiomers of the carboxylic acid amide is enantioselectivelyhydrolysed to form the corresponding enantiomerically enrichedcarboxylic acid, while the other enantiomer of the carboxylic acid amideremains unchanged.
 12. A process for the preparation of anenantiomerically enriched carboxylic acid and/or an enantiomericallyenriched carboxylic acid amide, which comprises contacting a mixture ofthe corresponding D- and L-carboxylic acid amides with the amidase ofclaim 10 in the presence of between 0.01 mM and 100 mM Zn²⁺, whereby oneof the enantiomers of the carboxylic acid amide is enantioselectivelyhydrolysed to form the corresponding enantiomerically enrichedcarboxylic acid, while the other enantiomer of the carboxylic acid amideremains unchanged.